Disclosure of Invention
Technical problem to be solved
Aiming at the defects of the prior art, the invention provides a recombinant mouse anti-human interleukin 19 monoclonal antibody, a preparation method and application, and the recombinant antibody has the advantages of definite gene structure, strong specificity, high titer and good stability, shows high sensitivity in various detection methods, and is suitable for being applied to in vitro diagnostic reagents.
(II) technical scheme
In order to achieve the purpose, the invention is realized by the following technical scheme: a recombinant mouse anti-human interleukin 19 monoclonal antibody, a preparation method and an application thereof comprise a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, wherein the light chain variable region comprises CDR1, CDR2 and CDR3, the coding gene sequence of the light chain variable region is sequentially shown in SEQ ID NO.1-3, the translated amino acid sequence of the light chain variable region is sequentially shown in SEQ ID NO.4-6, the heavy chain variable region comprises CDR1, CDR2 and CDR3, the coding gene sequence of the heavy chain variable region is sequentially shown in SEQ ID NO.7-9, and the translated amino acid sequence of the heavy chain variable region is sequentially shown in SEQ ID NO. 10-12.
The invention also discloses an expression vector containing the heavy chain variable region CDR1, CDR2 and CDR3 encoding genes.
The invention also discloses an expression vector containing the light chain variable region CDR1, CDR2 and CDR3 encoding genes.
The invention also discloses a preparation method of the recombinant mouse anti-human interleukin 19 monoclonal antibody, which is characterized by comprising the following steps: the method specifically comprises the following steps:
s1, immunizing a mouse with human interleukin 19 antigen to obtain a mouse splenocyte, fusing the mouse splenocyte with SP20 cells to obtain a hybridoma cell, extracting total RNA, and performing reverse transcription to obtain cDNA;
s2, using cDNA as a template to carry out primer design and amplify coding genes of a heavy chain variable region and a light chain variable region of a corresponding antibody;
s3, transferring the heavy chain variable region coding gene into an expression vector containing a heavy chain constant region gene, transferring the light chain variable region coding gene into an expression vector containing a light chain constant region gene, completing plasmid recombination, transferring the recombined plasmid into a competent cell, performing PCR amplification identification, and then arranging sequencing.
S4, carrying out amplification culture on the bacterial liquid corresponding to the plasmid with correct identification and sequencing, extracting the plasmid, co-transfecting the plasmid carrying heavy chain and light chain genes to mammalian cells, collecting the cultured cell supernatant, and carrying out protein purification to obtain the recombinant mouse anti-human interleukin 19 antibody.
Preferably, the specific steps of primer design in the amplification of the recombinant mouse anti-human interleukin 19 antibody variable region in step S3 are as follows: through multiple sequence alignment and degenerate primer design, primers capable of amplifying variable region genes of the recombinant mouse anti-human interleukin 19 antibody are designed in the relative constant regions of the leader peptide and the variable region.
Preferably, the sequence amplification in the amplification of the variable region of the recombinant mouse anti-human interleukin 19 antibody in step S3 specifically comprises the following steps: using cDNA as template, amplifying variable region gene by polymerase chain reaction, and identifying by agarose gel electrophoresis.
Preferably, the protein purification in step S4 specifically includes the following steps:
e1, purifying PCR products through a gel recovery kit, respectively connecting the heavy chain and light chain variable region gene segments with a seamless carrier, and transforming DH5 alpha competence;
e2, selecting four monoclonals for each adaptor, carrying out colony PCR by using universal primers, carrying out the size of a target fragment of 400-600, and selecting a positive clone bacterium with a bright band to send to a gene company for sequencing.
The invention also discloses an application of the recombinant mouse anti-human interleukin 19 monoclonal antibody in an in vitro diagnostic reagent.
(III) advantageous effects
The invention provides a recombinant mouse anti-human interleukin 19 monoclonal antibody, a preparation method and application. Compared with the prior art, the method has the following beneficial effects:
(1) the recombinant mouse anti-human interleukin 19 monoclonal antibody comprises a heavy chain constant region, a heavy chain variable region, a light chain constant region and a light chain variable region, wherein the light chain variable region contains CDR1, CDR2 and CDR3, the coding gene sequence of the light chain variable region is sequentially shown in SEQ ID NO.1-3, the translated amino acid sequence of the light chain variable region is sequentially shown in SEQ ID NO.4-6, the heavy chain variable region contains CDR1, CDR2 and CDR3, the coding gene sequence of the heavy chain variable region is sequentially shown in SEQ ID NO.7-9, and the translated amino acid sequence of the heavy chain variable region is sequentially shown in SEQ ID NO. 10-12. The unit production cost is low.
(2) The recombinant mouse anti-human interleukin 19 monoclonal antibody, the preparation method and the application have high recognition rate and good specificity on the epitope of human interleukin 19(IL19), and can well recognize natural antigens and recombinant antigens in tissues.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1 to 8, an embodiment of the present invention provides a technical solution: a recombinant mouse anti-human interleukin 19 monoclonal antibody, a preparation method and an application thereof, the source of biological materials is as follows: mouse spleen cells (Wuhan Baijiekang Biotechnology Co., Ltd.), SP2/0 (Wuhan Baijiekang Biotechnology Co., Ltd.), a mouse heavy chain constant region vector pBE27 (Wuhan Baijiekang Biotechnology Co., Ltd.), a vector pET28a (+) (Hunanfenghui Biotechnology Co., Ltd.), a mouse light chain constant region vector pBE28 (Wuhan Baijiekang Biotechnology Co., Ltd.), and other biological materials and reagents are all directly purchasable commodities.
Recombinant mouse anti-human interleukin 19(IL19) antibody variable region gene fishing
1. RNA extraction and reverse transcription of hybridoma cells to obtain cDNA
Firstly, immunizing a mouse by using human interleukin 19(IL19) antigen to obtain mouse spleen cells, fusing the mouse spleen cells with SP20 to obtain hybridoma cells, extracting hybridoma cell RNA by using a Triz1 method, and obtaining clear 28 and 18S bands through agarose gel electrophoresis, wherein the result of RNA integrity is better, the result of RNA concentration and purity measurement is D (260nm)/D (280nm) ═ 1.90, the requirement of the experiment can be met, reverse transcription is performed by using RNA as a template to synthesize cDNA, cDNA is used as a template, and a reference gene GAPDH in the mouse is used as a primer for PCR amplification to obtain a target band with the length of 320bp, and the reverse transcription cDNA can be used for subsequent experiments, and the reference figures 1-2 show that the reverse transcription cDNA can be used for the total RNA (A) and the cDNA amplification gel electrophoresis (B) of the hybridoma cells fused with the mouse spleen cells and SP 20.
2. Recombinant mouse anti-human interleukin 19(IL19) antibody variable region amplification
Designing a primer: through multiple sequence alignment and degenerate primer design, primers capable of amplifying variable region genes of the recombinant mouse anti-human interleukin 19(IL19) antibody are designed in the relative constant regions of the leader peptide and the variable region.
And (3) sequence amplification: using cDNA as template, amplifying variable region gene by polymerase chain reaction, and identifying by agarose gel electrophoresis, the result is shown in figure 3 and figure 4: the length of the heavy chain variable region gene is 336bp, the length of the light chain variable region gene is about 633bp, and the length of the heavy chain variable region gene is consistent with that of a target fragment.
3. Purification of variable region Gene fragment and ligation with pET28a (+) vector
PCR products are purified by a gel recovery kit, heavy chain and light chain variable region gene fragments are respectively connected with a seamless vector to transform DH5 alpha competence, 4 single clones are selected from each connector, colony PCR is carried out by using a universal primer, the size of a target fragment is about 400-600 (figure 5), and 1 positive clone bacterium with bright bands is selected and sent to a gene company for sequencing.
4. Sequencing result of variable region sequence of recombinant mouse anti-human interleukin 19(IL19) antibody
4.1 sequencing of the light chain variable region is shown below.
Gacattgtgctgacccagtctccagcttctttggctgtgtctctagggcagagggccaccatctcctgcaaggccagccaaagtgatgattatgctgtttatagttatatgaactggtaccaacagaaaccaggacagccacccaaactcctcatctatgctgcagccaatctagaatctgggatcccagccaggtttagtggcagtgggtctgggacagacttcatcctcaacatccatcctgtggaggaggaggatgttgcaacctattactgtcagcaaagtcatgaggatccgtacgcgttcggaggggggaccaagctggaaataaaac
The light chain genes were compared for homology using IMGT/QUEST online analysis software and the effective sequences are shown in Table 1 below:
TABLE 1 efficient sequence Listing for homology comparison of light chain genes
The encoding genes of the recombinant mouse anti-human interleukin 19(IL19) antibody and the light chain variable region CDR1, CDR2 and CDR3 are shown as SEQ ID NO: 1-3.
CDR1:caaagtgatgattatgctgtttatagttat
CDR2:gctgcagcc
CDR3:cagcaaagtcatgaggatccgtacgcg
The coding genes of the light chain variable region CDR1, CDR2 and CDR3 are translated into amino acid sequences which are sequentially shown as SEQ ID NO: 4-6.
CDR1:QSDDYAVYSY
CDR2:AAA
CDR3:QQSHEDPYA
4.2 heavy chain variable region sequencing results are shown below:
Caggtccagctgcaggagtctggacctgagctggtgaagcctggggcttcagtgaagatatcctgcaaggcttctggttactccatcactggctactacgtgcactgggtgaagcaaagccatgtaaggagccttgagtggattggacgtattaatccttaccatggcgctactaactacaaccagaatttcagggacaaggccagcttgactgtagataagtcctccaacacagcctacatggagctccacagcctgacatctgaggactctgcagtctattactgtgtctattgtaactactggggccaagggaccacggtcaagggcaattcc
the homology comparison of the heavy chain gene is carried out by using IMGT/QUEST online analysis software, and the effective sequence is shown in the following table 2:
TABLE 2 efficient sequence Listing for homology comparison of heavy chain genes
·(a)Other possibilities:Musmus_IGHJ3*01(highest number ofconsecutive identical nucleotides)。
The recombinant mouse anti-human interleukin 19(IL19) antibody has the coding genes of heavy chain variable region CDR1, CDR2 and CDR3 shown in sequence as SEQ ID NO: 7-9.
CDR1:ggttactccatcactggctactac
CDR2:attaatccttaccatggcgctact
CDR3:gtctattgtaactac
The coding genes of the heavy chain variable region CDR1, CDR2 and CDR3 are translated into amino acid sequences which are sequentially shown as SEQ ID NO: 10-12.
CDR1:GYSITGYY
CDR2:INPYHGAT
CDR3:VYCNY
Secondly, eukaryotic expression purification of recombinant mouse anti-human interleukin 19(IL19) antibody
Analyzing the sequenced sequence, selecting correct and functional heavy chain and light chain variable region genes, designing a second set of primers according to the sequencing result, carrying out 2 times of PCR by taking the previously sequenced genes as a template, using restriction enzymes, enzyme digestion PCR products, pBE27 and pBE28 to complete plasmid recombination, transferring into DH5 alpha competent cells, selecting positive clones and sequencing. Amplifying and culturing a bacterial liquid corresponding to the plasmid with correct sequencing, extracting the plasmid, co-transfecting the plasmid carrying heavy chain and light chain genes into 293F cells or CHO-S cells of mammal cells according to a certain proportion, collecting culture cell supernatant for 4-5 days, and purifying by protein A/G affinity chromatography, wherein the result of SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) electrophoretic analysis of the purified antibody is shown in figure 6, and the purified antibody is the recombinant mouse anti-human interleukin 19(IL19) antibody.
Application of recombinant mouse anti-human interleukin 19(IL19) antibody activity detection
3.1Western blot detection: the result of using a recombinant mouse anti-human interleukin 19(IL19) antibody with a final concentration of 2ug/ml as a primary antibody and a secondary antibody as goat anti-mouse IgG (using a concentration of 0.1ug/ml) and detecting the sample as human milk and recombinant human interleukin 19(IL19) protein is shown in fig. 8, wherein lane a is human milk and lane B is recombinant human interleukin 19(IL19) protein, and the result shows that the recombinant mouse anti-human interleukin 19(IL19) antibody can well identify a natural sample (25kd) and a prokaryotic expression recombinant protein (20 kd).
3.2ELISA Titers assay: the recombinant human interleukin 19(IL19) (2 mu g/ml) is used for coating the enzyme label plate, after 1% BSA is used for sealing and incubation, the recombinant mouse anti-human interleukin 19(IL19) monoclonal antibody is diluted from 1 mu g/ml according to 2 times of gradient to form 13 gradients, the diluted recombinant antibody is added into the coated enzyme label plate, and the result shows that the recombinant mouse anti-human interleukin 19(IL19) monoclonal antibody and the recombinant human interleukin 19(IL19) can be specifically combined and have better dose-effect relationship, the result is shown in Table 2, and the EC50 value of the recombinant mouse anti-human interleukin 19(IL19) monoclonal antibody is 0.021 mu g/ml by curve fitting in figure 7.
Table 2ELISA test the binding potency of recombinant mouse anti-human interleukin 19(IL19) monoclonal antibody to recombinant human interleukin 19(IL 19).
Sequence listing
Light chain sequence
CTAACACTCATTCCTGTTGAAGCTCCTGACAATGGGTGAAGTTGATGTCTTGTGAGTGGCCTCACAGGTATAGCTGTTATGTCGTTCATACTCGTCCTTGGTCAACGTGAGGGTGCTGCTCATGCTGTAGGTGCTGTCTTTGCTGTCCTGATCAGTCCAACTGTTCAGGACGCCATTTTGTCGTTCACTGCCATCAATCTTCCACTTGACATTGATGTCTTTGGGGTAGAAGTTGTTCAAGAAGCACACGACTGAGGCACCTCCAGATGTTAACTGCTCACTGGATGGTGGGAAGATGGATACAGTTGGTGCAGCATCAGCCCGCTTGATTTCCAGCTTGGTGCCTCCACCGAACGTCGGAGGAATACCATAATGATGTTGACAGTGATAATTCCCAAAATCTTCAGGCTTCAGGTTGTTGATCTTCAGAGAAAACTGTGTGCCTGATCCACTGCCACTGAACCTTGATGGCACACCTTCTGTTAAGGTTTTTGCATTATAGACCAGGAGCTGAGGAGATTTTCCCTGCTTCTGCTGATACCATGCTAAATAACTGTAAATATTCTCACTTGTTCGACATGTGATGGTGACAGTTTCTCCCACAGATGCAGATAGGGAGGCTGGAGACTGAGTCAGCTGGATGTC
Heavy chain sequence
AGAGTTCCAGGTCACTGTCACTGGCTCAGGGAAATAGCCCTTGACCAGGCATCCCAGGGTCACCATGGAGTTAGTTTGGGCAGCAGATCCAGGGGCCAGTGGATAGACAGATGGGGGTGTCGTTTTGGCTGAGGAGACGGTGACTGAGGTTCCTTGACCCCAGTAGTCCATAGTATAGTCGTAGCGACTACCGTAGTGAGACCATCTTGCACAGAAATAGATTGCAGAGTCCTCAGATGTCAGGCTGCTGAGCTGCATGTAGGCTGTGCTGGAGGATGTGTCTGAAGTCAGTGTGGCCTTGCCCTTGAACTTCTCATTGTATTCAGGATAACCATTTCCAATATAAATATATCCAATCCATTCCAGGCCCTGTCCAGGCCTCTGTTTCACCCAGTTTATACCGTAGCTTGTGAATGTATATCCAGAAGTCTTGCAGGACATCTTCACTGAGGACCCAGGCCTCACCAGCTCAGCTCCAGACTGCTTCAGCTGCACCTG。
Compared with the prior art, the antibody has definite protein and gene structures and good stability, can prepare a new cell strain capable of expressing the antibody again even if a cell strain expressing the antibody does not exist, has small batch difference, high antibody titer, high antibody yield and low unit production cost, has high recognition rate and good specificity on the epitope of the human interleukin 19(IL19), and can well recognize both natural antigen and recombinant antigen in tissues.
And those not described in detail in this specification are well within the skill of those in the art.
It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.
Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.